1. Introduction
Titanium and its alloys are strong, lightweight metals that are highly durable, corrosion-resistant, and biocompatible. In dentistry, implants made from titanium-based materials serve as solutions for replacing missing teeth and even reconstructing oral cancer defects. Titanium dental implants possess the unique ability to osseointegrate, especially after appropriate surface treatments such as SLA (sandblasted with large-grits and acid-etched), plasma-spray, and nanotubes. These treatments enable a stable and long-lasting foundation for prostheses, including crowns, bridges, and dentures [
1]. Overall, dental implants not only improve the patient’s function and aesthetics, but also enhance their overall oral health and quality of life [
2].
Candida albicans, a common opportunistic fungal pathogen, can pose a potential risk to the success of titanium dental implants. This microorganism is known to cause oral infections, particularly in individuals with weakened immune systems or underlying health conditions [
3]. When
Candida albicans colonizes the surface of a titanium dental implant, it forms a biofilm—a complex structure of microorganisms embedded within an extracellular matrix. This biofilm not only provides protection to the fungal cells from the host’s immune response and antifungal treatments, but also increases the likelihood of implant-associated infections [
4]. The occurrence of
Candida albicans growth on titanium surfaces is significant. An in vivo study indicated a 75% incidence of
Candida albicans growth on cp titanium [
5]. Consequently, the presence of
Candida albicans on titanium dental implants may compromise the implant’s stability and longevity, leading to complications such as peri-implantitis, implant failure, or the need for additional dental interventions. Researchers are actively exploring strategies to prevent Candida albicans colonization on titanium dental implants to ensure optimal oral health and implant success [
6,
7,
8].
To minimize the risk of implant failure and reduce the likelihood of complications such as infection or inflammation, various strategies have been investigated to prevent
Candida albicans colonization on titanium dental implants. One approach involves modifying the implant surface with antimicrobial agents, such as silver nanoparticles (AgNPs), which exhibit potent antifungal properties. Research studies have demonstrated that incorporating AgNPs on the implant surface can significantly reduce
Candida albicans adhesion and biofilm formation, thereby mitigating the potential for implant-associated infections [
9]. Another approach includes the use of photodynamic therapy (PDT), a non-invasive technique that employs light-activated photosensitizers to generate reactive oxygen species (ROS) capable of destroying fungal cells [
10]. Additionally, the application of antifungal agents such as fluconazole and chlorhexidine has been shown to inhibit
Candida albicans growth on titanium surfaces [
11]. However, these approaches may only be effective in the short-term under specific conditions, and could potentially induce antifungal resistance. Thus, a newer approach is necessary.
Silanes possess versatile coupling capabilities and are commonly used as coupling agents to form stable covalent bonds on titanium dental implants with resin cements. Silanes can be classified as functional (C=C) and non-functional (alkoxy) silanes based on their existing functional groups. The functional group 3-(trimethoxysilyl)propyl methacrylate (γ-MPS) is the most widely used in silane coupling agents, offering ideal adhesive properties and interfacial stability [
12]. Other functional groups, such as 3-acryloxypropyltrimethoxysilane (ACPS) [
13], quaternary ammonium salt (QAS) [
14], vinyltrimethoxysilane [
15], 3-isocyanato-propyltriethoxysilane (IPTES) [
16], 3-aminopropyltriethoxysilane (APTES) [
17], urethane dimethacrylate silane (UDMS) [
18], 3-glycidoxypropyltrimethoxysilane (GPS) [
19], and styrylethyltrimethoxysilane (SETMS) [
20], have also been extensively studied in the dental field.
Non-functional silanes, which have a hydrolysable alkoxy group, can also be used as silane coupling agents. In certain scenarios, non-functional silanes can possess dual Si elements, with each containing three alkoxy groups capable of creating tight cross-linking networks within resin–titanium adhesion [
21]. The typical non-functional silanes that act as cross-linkers include bis-1,2-(triethoxysilyl)ethane (BTSE) [
22], bis-[3-(trimethoxysilyl)propyl]amine [
23], bis-1,6-(trichloroxysilyl)hexane (BISHEX) [
24], and bis-1,8-(trichloroxysilyl)octane (BISOCT) [
24].
The dual silanization technique involves blending functional and non-functional silanes in varying mass ratios, which results in a highly adhesive interface between titanium and resin cement. This is accomplished by leveraging the high reactivity of the functional group and the hydrophobicity of the non-functional group. Dual silanization enhances bonding by increasing double bond conversion, reducing water sorption, and minimizing polymerization stress [
21].
Villard et al. [
25] found that the commercially available γ-MPS and experimental dual silane blend ACPS and BTSE can effectively reduce
Candida albicans adhesion on sandblasted titanium, with the blended silane showing better resin–titanium adhesion strength. A study conducted by Nikawa et al. [
14] demonstrated that a titanium surface coated with silane containing quaternary ammonium salts (QASs) exhibited a substantial reduction in
Candida albicans adhesion compared to uncoated titanium surfaces. This silane-based QAS coating effectively disrupted the fungal cell membrane, resulting in an enhanced antifungal efficacy. In terms of osteogenesis, silane did not impair bone formation, but even increased the bone-to-implant contact on SLA-Ti surfaces. Thus, utilizing silanes as a method to modify titanium dental implant surfaces offers a promising strategy to prevent
Candida albicans colonization. A dual silanization primer has been designed and accepted unanimously as a dental primer that promotes adhesion effectively between dissimilar materials. As such a silane primer is designed for in vivo use, apart from its adhesive properties, its antimicrobial properties are also worth investigating. However, very few studies have investigated the antimicrobial properties of this type of material.
In this study, the Candida albicans colonization effects of the blended silane primer on SLA-Ti surfaces were evaluated, with SLA-Ti without silanization serving as a positive control and a flat titanium surface as a negative control. The null hypothesis was that there would be no significant differences in the Candida albicans colonization effects on all the titanium surfaces.
2. Materials and Methods
2.1. Titanium Discs
In this study, 18 SLA-Ti discs (Institute Straumann AG, Basel, Switzerland) with a diameter of approximately 15 mm were used, and 9 grade 2 cp-Ti discs (ASTM B265, Daido Steel Co. Ltd., Aichi, Japan) with a diameter of approximately 9.79 mm were used. The cp-Ti discs were polished by 4000-grit SiC abrasive paper before use.
2.2. Silane Preparation
The silane primer was prepared according to the procedures of Matinlinna and co-workers [
26], using a blended silane with a concentration of 1.0 vol% 3-ACPS and 0.3 vol% BTSE (
Figure 1). In brief, a solvent of 95% ethanol was first mixed in deionized (DI) water (Milli-Q water; resistivity: 18.2 MΩ cm) in a volumetric ratio of 95:5 (pH adjusted to 4.5 by adding 1.0 M acetic acid) in a polypropylene (PP) bottle. This acidified solvent was then stabilized for 24 h at room temperature and refrigerated at 4 °C.
Next, 30 μL of BTSE (Gelest Inc., Morrisville, PA, USA) was added to 4.97 mL of the acidified solvent, mixed well, and left to stabilize for one hour. After that, 100 μL of ACPS (Gelest Inc., Morrisville, PA, USA) was added to the 4.9 mL acidified solvent. This 5.0 mL of 1.0 vol% ACPS and 5.0 mL of 0.3 vol% BTSE were then hand-mixed thoroughly by shaking in a plastic centrifuge tube for 1 mm. The 10 mL of blended silane primer was then wrapped entirely with aluminum foil and stored in a 4 °C refrigerator. After another hour of activation, the silane primer was ready to be applied to the specimens. This silane primer was freshly prepared for each use. Fourier-transform infrared spectroscopy (FTIR) was used to confirm the effectiveness of the prepared silane.
2.3. Silanization of Specimen Surface
Nine SLA-Ti discs were silanized using the prepared silane mentioned in the above section. The experimental silane solution was applied onto the surface of each SLA-Ti disc manually. Each sample was brushed twice, with a new brush each time, by the same operator. The brushing process was identical to clinical operations, with a gentle brushing force of around 1 N. The whole procedure was performed under aseptic conditions using a flame sterilization technique. After silanization, the specimens were left for 5 min at room temperature in sterile 24-well plates (Iwaki, Tokyo, Japan) to allow the silanes to react and set onto the surfaces. Subsequently, the surface was characterized by energy-dispersive X-ray spectroscopy (EDX) (IXRF Systems, Inc., Austin, TX, USA).
2.4. Microbiology
2.4.1. Sample Allocation, Pretreatment, and Post-Treatment
Prior to culturing, two discs from each of the control groups underwent pre-cleaning in an ultrasonic bath (Decon FS200; Decon Ultrasonics Ltd., Hove, UK) according to the following specified cleaning protocol [
6]: (i) immersion in 95% ethanol for 5 min; (ii) rinsing with deionized (DI) water for 3 min; and (iii) exposure to acetone for 5 min. Then, the specimens were washed with DI water and subsequently subjected to steam autoclaving (Autoclave ASB300BT, ASTELL, London, UK) at 121 °C for 15 min. The sterilized specimens, along with the silanized SLA-Ti discs, were then arranged in sterile 24-well plates (Iwaki, Tokyo, Japan).
2.4.2. Culturing of Candida albicans and Biofilm Development
Candida albicans (ATCC 90028) was incubated on Sabouraud dextrose agar (Gibco, Paisley, UK) at 37 °C for 18 h. The cells were then washed twice in phosphate-buffered saline (PBS) at a pH of 7.2, and the resulting pellet was collected through centrifugation at 4000 rpm for 10 min using a centrifuge (GS-15R, Beckman Instruments Inc., Brea, CA, USA). A cell suspension was prepared using 100 mM glucose and yeast nitrogen base (Difco Laboratory Inc., Detroit, MI, USA) at McFarland scale 4. Following the standardization of the culture, 1.5 mL of the cell suspension was applied to ensure the complete coverage of the material surfaces. The plate was placed in a shaking incubator (Stuart SI500, Bibby Scientific Ltd., Staffordshire, UK) at 37 °C and 80 rpm for 90 min. Subsequently, the media were replaced, and the plate was further incubated for 48 h, with media changes every 24 h. After 48 h of biofilm growth, the specimens were rinsed in PBS and transferred to new plates with 1.0 mL of PBS per well. The attached biofilm on each specimen was then removed by repeated pipetting.
2.4.3. Colony Forming Units (CFUs)
In this research, the cell suspension collected from each well was diluted by factors of 10
−2, 10
−3, and 10
−4. Subsequently, 50 μL of each diluted cell suspension was spread on Sabouraud agar using a spiral plater (Autoplate 4000; Spiral Biotech Inc., Bethesda, MD, USA). The agar plates were then incubated at 37 °C for 48 h. The purpose of employing different dilution factors was to generate plates with colony counts ranging from 30 to 300. If the counts fell outside this range, counts from an alternate dilution factor would be utilized. The calculation of CFU/mL for each group was conducted using the formula:
The volume plated was 0.05 mL. Additionally, considering that each specimen may have varying diameters, the results presented are per unit area (mm2). Three trials were performed for each group.
2.4.4. Real-Time Polymerase Chain Reaction
For the real-time polymerase chain reaction (RT-PCR) analysis, initially the DNA of Candida albicans was extracted by following the procedure of a DNA extraction kit. This involved harvesting cells from each sample through centrifugation (Sorvall Legend Micro 21 centrifuge; Thermo Electron LED GmbH., Osterode, Germany) at the maximum speed (14,500 rpm) for 1 min. Subsequently, the supernatant was removed, and the solid residues were resuspended in 293 μL of 50 mM ethylenediaminetetraacetic acid in an Eppendorf tube. A 15 μL lyticase (20 mg/mL) was then added to each tube, followed by incubation in a water bath (Jalabo TW12; Jalabo Labortechnik GmbH., Seelbach, Germany) at 37 °C for 1 h. After centrifuging the tubes at 13,000 rpm for 5 min and discarding the supernatant, the spheroplasts were resuspended in 180 μL of tissue lysis buffer (Buffer ATL; QIAGEN GmbH, Hilden, Germany). A total of 20 μL of proteinase K was added to each tube, and the tubes were then vortexed and incubated in a water bath at 56 °C for 10 min. This was followed by the addition of 200 μL of lysis buffer (Buffer AL; QIAGEN GmbH., Hilden, Germany), vortexing for 15 s, and further incubation in a water bath at 70 °C for 10 min. A total of 200 μL of absolute ethanol was carefully applied to spin columns (QIAGEN GmbH., Hilden, Germany), and the columns were centrifuged at 8000 rpm for 1 min. The process continued with the addition of wash buffer 1 (Buffer AW1; QIAGEN GmbH., Hilden, Germany) and wash buffer 2 (Buffer AW2; QIAGEN GmbH., Hilden, Germany) to the columns, followed by the final elution step in new 1.5 mL Eppendorf tubes with 100 μL of elution buffer (Buffer AE; QIAGEN GmbH., Hilden, Germany), and centrifugation at 8000 rpm for 1 min.
For the post-DNA extraction procedures, the DNA of Candida albicans was collected and stored in Eppendorf tubes, referred to as the “cell solution”, at −21 °C until the RT-PCR analysis. Simultaneously, a “master mix” solution was prepared, containing 5 μL of a nucleic acid stain (QuanitFast SYBR Green; QIAGEN GmbH., Hilden, Germany), 2 μL of DI water, 1 μL of forward primer (5′ GGG TTT GCT TGA AAG ACG GTA 3′), and 1 μL of reverse primer (5′ TTG AAG ATA TAC GTG GTG GAC GTT 3′).
To quantify the amount of Candida albicans using RT-PCR, standard curves were initially generated by utilizing known cell-concentration solutions with varying counts of Candida albicans cells. The standard curves were generated through StepOne Software, V2.2. Following this, separate wells of a 0.1 mL well plate (MicroAmp Fast optical 96-Wellplate; Applied Biosystems Pty Ltd., Scoresby, Australia) were used to mix the “cell solution” with the “master mix”. The plate was covered with a cohesive cover (MicroAmp optical adhesive film; Applied Biosystems Pty Ltd., Scoresby, Australia) and analyzed using a PCR machine (StepOnePlus; Applied Biosystems Pty Ltd., Scoresby, Australia). To ensure the result accuracy and reliability, each group underwent the analysis six times.
2.5. Surface Morphology
The 3D surface topography images and surface roughness of the titanium samples from three groups were investigated with atomic force microscopy (AFM, Dimension Edge, Bruker, Billerica, MA, USA) using the contact mode for measurements. Scanning electron microscopy (SEM) was used to visualize the biofilm; the specimens were first fixed by immersing them in 2.5% glutaraldehyde (BDH Lab. Supplies, Poole, UK) for 1.5 h. Following fixation, the samples were dehydrated through a series of ethanol solutions, including 70%, 85%, and 95%, for 15 min each, and then immersed in absolute ethanol twice for 15 min each time. Once dehydrated, the dry specimens were mounted on aluminum stubs and sputter-coated with a Pd-Pt coating. Finally, the samples were observed using SEM (SU-1510, Hitachi, Tokyo, Japan) at an acceleration voltage of 15 kV in high-vacuum mode.
2.6. Statistical Analysis
The statistical analysis was performed for the CFU and PCR results using the SPSS software (version 25, IBM, Newark, NJ, USA). The Kruskal–Wallis test and Mann–Whitney U test were used to determine the statistical significance at an α level of 0.05.
4. Discussion
In this study, the antimicrobial activity of ACPS-BSTE dual silane against the early stage of Candida albicans biofilm formation was evaluated. The Candida albicans biofilm was successfully cultured on all the cp-Ti, SLA-Ti, and silane-coated SLA-Ti surfaces. This finding might suggest that, if such titanium alloys and surfaces are utilized for dental implants, there is a possibility of biofilm formation on the surface. The silane-coated SLA-Ti demonstrated a significantly lower CFU count compared to the non-coated SLA-Ti. However, no such significance was observed according to the RT-PCR results. Consequently, the null hypothesis was partially rejected. Based on the findings of this study, ACPS and BTSE can kill fungi on titanium surfaces, but they do not have a prominent effect in preventing fungal attachment.
The findings of the present study indicate that silanized SLA-Ti surfaces exhibited a lower CFU than non-silanized SLA-Ti or cp-Ti. Nonetheless, the silanized SLA-Ti surface displayed a high Ct value in the RT-PCR results. Both RT-PCR and CFU are methodologies employed to quantify microbial adhesion, but they operate on different principles and measure the distinct parameters and performance of the biological sample, which may contain live or dead cells. The CFU quantifies live microbes by culturing Candida albicans on an agar plate, while RT-PCR measures DNA fragments of cells, irrespective of their viability. Consequently, RT-PCR detects the total cell number of both live and dead microbes, while the CFU detects only the live cell number. In this study, the CFU analysis revealed that silanization can significantly reduce the adherence of live microbes on SLA-Ti surfaces. However, RT-PCR demonstrated no significant difference among all three groups, with the silane-coated SLA-Ti surface exhibiting the highest mean microbe count, while the other two groups displayed very similar mean microbe counts.
In this study, commercially available SLA-Ti (Institute Straumann AG, Basel, Switzerland) was utilized as the substrate material. SLA, which stands for “sandblasted, large-grits, and acid-etched”, is a technique that involves large-grit sandblasting to create macro-roughness on the titanium surface, followed by acid etching that superposes a micro-roughness. The resulting topography provides an ideal structure for cell attachment [
27]. However, the increased macro- and micro-roughness, while promoting cell attachment, also enhances microbial colonization. If left uncleaned, this can lead to peri-implantitis, which damages alveolar bone and connective tissues, ultimately resulting in implant failure [
28,
29]. Therefore, although the etched micro-roughness surface promotes bone cell growth, proper cleaning and maintenance are crucial to prevent microbial colonization and potential implant failure.
The change in the surface free energy of silanized SLA-Ti surfaces can also impact microbial adhesion. In an in vitro study by Burgers et al., it was found that surface free energy is more critical than surface roughness in the initial adhesion of
Candida albicans to titanium and zirconia implant surfaces [
30]. A positive correlation exists between surface roughness and surface free energy: Increased surface micro-roughness can expand the surface area and enhance the surface free energy [
31]. Furthermore, most oral microorganisms possess a high surface free energy at the cell surface and, as a result, prefer to bind to surfaces with a high surface free energy [
32]. The threshold for surface free energy is 50 mJ/m
2, and oral microorganisms with a surface free energy lower than 50 mJ/m
2 are more challenging to attach [
33]. Teughels et al. also discovered that a lower surface free energy significantly negatively impacted biofilm formation on coated surfaces [
34].
Dental implants can be classified as screw-retained or cement-retained. In clinical practice, cement-retained implants are more commonly used due to their superior handling properties [
35], aesthetic performance, and lower cost of fabrication. Ideally, when cementing a cement-retained implant, the cement should not come into contact with soft tissues. However, in reality, up to 50 times the ideal amount of cement may be placed, leading to excess cement being exuded onto the gingiva [
36]. This can increase the risk of microbial colonization and biofilm formation around the abutment–cement and cement–crown interface, which can contribute to implant failure.
Moreover, during the implant placement process, the implant can come into contact with microbes present in the oral cavity. Incorporating antimicrobial properties into the silane coupling agent can help prevent microbial colonization and biofilm formation, even in areas that are not directly exposed to the oral cavity or external environment. This can reduce the risk of implant failure and improve overall patient outcomes.
Matinlinna et al. [
13] introduced a new dual silane consisting of ACPS and BSTE. The concentrations of 1.0 vol% ACPS and 0.3 vol% BTSE in the blend were found to be the most optimized concentrations. This ratio has been demonstrated to significantly improve the shear bond strength values of a resin cement bond with titanium and ceramics [
15,
22,
26]. This study also found that the shear bond strength between resin cement and zirconia had a negative correlation with the number of thermal cycles increasing under aging conditions. Furthermore, ACPS exhibited a significantly higher shear bond strength compared to γ-MPS, of up to 8000 cycles [
13,
37].
Most studies on the antimicrobial properties of the Ti–silane interface have been focused on the addition of antimicrobial elements to titanium, such as Ag, Al, and Cu [
6,
38,
39]. Alternatively, on the silane side, quaternary ammonium salts (QASs) have also been found to have good antimicrobial properties, as they kill microbes through electrostatic adsorption and insertion into cell membranes, which can alter the cell membrane permeability and reduce the probability of microbes developing drug resistance. Several studies have investigated the use of a QAS coating on biomaterials to control microbial adhesion [
40,
41]. QASs were first reported in the 1970s [
40], and the alkyl chain length of different ion combinations (Cl
−, Br
−, and I
−) has been found to be highly linked to the antimicrobial activity against both Gram-positive and Gram-negative microbes, with the counterion (Cl
−, Br
−, and I
−) affecting the antimicrobial activity of the other Gram-negative microbes, yeasts, and fungi. Coupling agents with a C10 alkyl chain and Cl
− or Br
− counter-ions (10-Cl or 10-Br) have been found to have the highest antimicrobial activity due to the cationic and amphiphilic nature of the ammonium [
42].
The introduction of new elements may cause changes in interfacial properties and may cause allergies to the surrounding tissues. For example, QASs can cause allergic skin rashes even with limited exposure, making biocompatibility and water absorption two main concerns when used in the oral environment [
43]. Therefore, special care needs to be taken to ensure that the quaternary ammonium groups are tightly attached to the substrate to prevent the ammonium from leaking into the oral environment and creating antifungal effects in undesired areas. In contrast, ACPS and BSTE contain only C, H, O, and Si elements (
Figure 1), so their antifungal properties may not be as good as QASs. However, they do not encounter the abovementioned disadvantages. Further experiments are required to investigated the exact effective concentrations of ACPS and BTSE to inhibit and kill microorganisms as well as prolong the shelf-life.